1. Field of the Invention
The present invention relates to a method for producing L-isoleucine by fermentation, and particularly to a method for producing L-isoleucine with a microorganism of the genus Brevibacterium and Corynebacterium constructed by a gene splicing technique.
2. Description of the Prior Art
In the past, in order to render a wild strain capable of producing L-isoleucine from carbohydrates, it has been necessary to induce artificial mutants from the wild strain. In this regard there are many known L-isoleucine producing artificial mutants.
Examples of known isoleucine producing microorganisms include mutants of Serratia resistant to isoleucine-hydroxamate (Japanese Published Examined Patent Application No. 30593/1977), mutants of Corynebacterium glutamicum requiring L-leucine for growth (Japanese Published Examined Patent Application No. 38995/1972), mutants of Brevibacterium and Corynebacterium resistant to .alpha.-amino-.beta.-hydroxy valeric acid (hereinafter referred to as AHV), (Japanese Published Examined Patent Application No. 2880/1967), mutants of Brevibacterium resistant to AHV and requiring lysine for growth (Japanese Published Examined Patent Application No. 6237/1976), mutants of Brevibacterium resistant to AHV and O-methylthreonine (Japanese Published Examined Patent Application No. 21077/1976), mutants of Corynebacterium resistant to S-(2-aminoethyl)-cysteine (Japanese Published Unexamined Patent Application No. 61290/1977), mutants of Escherichia resistant to 2-amino-3-methyl thiobutyric acid (Japanese Published Unexamined Patent Application No. 69881/1978) and mutants of Brevibacterium resistant to AHV and trichloroalanine (Japanese Published Unexamined Patent Application No. 35287/1979).
Another approach to increase the productivity of amino acids in microorganisms is found in U.S. Pat. No. 4,278,765 and in Japanese Published Unexamined Patent Application Nos. 131397/1980, 1890/1981, 18596/1981, 82095/1981, 85287/1981, 117795/1981, 144092/1981 and 144093/1981. In this technique Escherichia coli stains transformed with a recombinant plasmid DNA and constructed by a gene splicing technique to produce various kinds of amino acids are disclosed.
However, it has been difficult to construct a commercially useful isoleucine producer of Escherichia coli by the gene splicing technique, because original Escherichia strains do not express high productivity for L-isoleucine and recombinant strains derived from such Escherichia strains do not product high amounts of L-isoleucine.
On the other hand, there are many strains in the genera of Brevibacterium and Corynebacterium which produce large amounts of L-isoleucine, and for this reason there may be strains of Corynebacterium and Brevibacterium which are suitable as original strains for the construction of L-isoleucine producers by the gene splicing technique. However, although the presence of plasmids in the strains of Brevibacterium and Corynebacterium are known (Publication of European Patent Application No. 0030391), the plasmids have no specific characteristics which would be useful as a marker for the identification of the plasmids. Therefore, it has been very difficult to select recombinant plasmids, derived from the plasmids of Brevibacterium and Corynebacterium. For the reason noted above, it has been difficult to construct an L-isoleucine producer from L-isoleucine producing Brevibacterium and Corynebacterium by the gene splicing technique.
A need, therefore, continues to exist for the development of a process for production of L-isoleucine in high yields.